The present invention generally relates to a method and apparatus for accurately decomposing images constructed from the fluorescent emissions of a plurality of fluorescent dyes coexisting in a specimen, with application to a broad range of imaging instruments and particularly, to flow imaging instruments using time delay integration image detectors and spectral dispersion in a single plane accomplished using a prism.
Fluorescent compounds are useful for labeling components of biological cells. Such labeling is useful for conducting basic research, as well as for medical diagnostic testing. Fluorescent compounds can facilitate the detection of molecular structures in cells. Cellular specimens are exposed to dyes in which molecules of one or more fluorophores are attached to molecules that bind with the target molecules in the cells. The binding mechanism may be an antigen-antibody interaction, or the hybridization of a target strand of nucleic acid with its complementary counterpart. In antigen-antibody interaction, the flourophore is attached to a protein complex. In the hybridization of a target strand, the fluorophore is attached to a strand of RNA or DNA of a particular base sequence.
Information about biological specimens stained with fluorescent markers can be collected by a variety of methods. Slides carrying cells or tissue sections may be viewed through a microscope equipped with the appropriate excitation sources and optical filters for the fluorophores in use. Alternatively, cells may be suspended in a liquid and passed through a flow cytometer equipped to detect and count cells displaying various bound fluorophores. A preferred flow imaging technology, utilizing time delay integration electronic image capture and computational image analysis to deliver information about the specimen, is disclosed in commonly assigned U.S. Pat. No. 6,211,955, the complete disclosure, specification, and drawings of which are hereby specifically incorporated by reference.
An extension of the technology of fluorescence flow imaging is that of utilizing multiple fluorescent dyes in a single specimen and separating the signals from the plurality of dyes by wavelength discrimination. While such multiplexed signals enable more information about each cell to be collected, the signal separation process can be challenging. It would be desirable to provide a signal separation process that accurately estimates the relative concentrations of fluorescent dyes bound to a specimen, and, therefore, about the relative abundances of a plurality of molecular species in the specimen. Such multiplexed information is especially valuable for characterizing the reactions of biological cells to compounds under investigation as potential therapeutic agents or for detecting abnormalities in genetic makeup or gene expression related to disease.
One aspect of the present invention is a method for processing an electrical signal containing data from at least two sources, to separate the electrical signal into components corresponding to each one of the at least two sources. Once the electrical signal containing data from at least two sources is obtained, it is processed to separate the electrical signal into portions corresponding to each one of the at least two sources. Once separated, amplitudes are derived for each portion of the electrical signal that corresponds one of the at least two different sources. Preferably each source is a fluorophore, and the amplitude for each fluorophore corresponds to a relative concentration for that fluorophore.
In one embodiment the electrical signal is processed using curve fitting to distinguish different portions of the electrical signal corresponding to different sources. The curve fitting can be achieved using Lorentzian equations.
A model of the spectra of the at least two sources is preferably obtained before processing the electrical signal. The step of using curve fitting to distinguish different portions of the electrical signal comprises the step of using nonlinear conjugation to reduce an error between the electrical signal and the model. More preferably, the step of using nonlinear conjugation is performed iteratively. In at least one embodiment, the error that is reduced by the nonlinear conjugation comprises a mean square error between the model and the electrical signal.
The electrical signal is preferably obtained by focusing light from an object including at least two sources along a collection path, and dispersing the light that is traveling along the collection path into a plurality of light beams, such that each light beam corresponds to a different wavelength. The plurality of light beams are focused to produce respective images for the light beams, and the images are directed to a detector, thereby generating the electrical signal.
In yet another embodiment, processing the electrical signal is achieved by solving a set of linear equations corresponding to an emission set defined by the at least two different sources.
Yet another aspect of the invention is directed to a method for determining a relative concentration of a specific fluorophore associated with an object that includes at least two different fluorophores. Light is focused from the object along a collection path. The light traveling along the collection path is dispersed into a plurality of light beams, such that each light beam corresponds to a different wavelength. A prism is preferably employed to achieve the dispersion. Each of the light beams is focused to produce a respective image, and the images are detected by a detector that produces an electric signal in response to the images. The electrical signal is processed to separate it into portions corresponding to each of the at least two different fluorophores. An amplitude is derived for each portion of the electrical signal that corresponds one of the at least two different fluorophores. The amplitude for each fluorophore corresponds to a relative concentration for that fluorophore.
The step of processing the electrical signal includes either using curve fitting to distinguish different portions of the electrical signal corresponding to different fluorophores from one another, or solving a set of linear equations based on an emission set defined by the different fluorophores represent in the electrical signal.
A model of the spectra of the at least two fluorophores is obtained before processing the electric signal, and the step of using curve fitting to distinguish different portions of the electrical signal includes the step of using nonlinear conjugation to reduce an error between the electrical signal and the model. Preferably, the nonlinear conjugation is performed iteratively. In at least one embodiment, the error that is reduced by the nonlinear conjugation comprises a mean square error between the model and the electrical signal.
In embodiments in which a set of linear equations is solved, the detector preferably comprises a scatter channel and a fluorescence channel. The step of solving a set of linear equations includes the steps of establishing a pixel positional reference for the scatter channel and the fluorescence channel of the detector, and determining a lateral shift in the fluorescence channel. The lateral shift is preferably determined with sub-pixel accuracy.
In addition to the aforementioned embodiments relating to the method, the present invention is also directed to a system having elements that carry out functions generally consistent with the steps of the method described above. Specifically, an imaging system is defined for determining a relative concentration of a specific fluorophore associated with an object. The imaging system includes a collection lens disposed so that light traveling from the object passes through the collection lens and travels along a collection path. A dispersing component is disposed in the collection path so as to receive the light that has passed through the collection lens, dispersing the light into a plurality of separate light beams. Each light beam is directed away from the dispersing component in a different predetermined direction. The system also includes an imaging lens disposed to receive the light beams from the dispersing component, thereby producing an ensemble of images that includes a plurality of images corresponding to each of the light beams. Each image is projected by the imaging lens toward a different predetermined location. A detector is disposed to receive the plurality of images produced by the imaging lens, and produces an output signal in response thereto. Finally, the system includes means for processing the output signal to separate the electrical signal into portions corresponding to different fluorophores, and to derive an amplitude for each portion of the electrical signal that corresponds to different fluorophores. The amplitude for each fluorophore corresponds to a relative concentration for that fluorophore.
The means for processing preferably includes a memory in which a plurality of machine instructions defining a signal conditioning software program are stored, and a processor that is coupled to the display, and to the memory to access the machine instructions. Execution of the machine instructions by the processor causes it to separate the electrical signal into portions corresponding to different fluorophores, and to derive an amplitude for each portion of the electrical signal that corresponds to a different fluorophore.
It is contemplated that the means for processing the signal might comprise either a programmed computer, an application specific integrated circuit (ASIC), or an oscilloscope.